The invention relates to antisense IAP nucleic acids and methods of using them to increase apoptosis.
One way by which cells die is referred to as apoptosis, or programmed cell death. Apoptosis often occurs as a normal part of the development and maintenance of healthy tissues. The process may occur so rapidly that it is difficult to detect.
The apoptosis pathway is now known to play a critical role in embryonic development, viral pathogenesis, cancer, autoimmune disorders, and neurodegenerative diseases, as well as other events. The failure of an apoptotic response has been implicated in the development of cancer, autoimmune disorders, such as lupus erythematosis and multiple sclerosis, and in viral infections, including those associated with herpes virus, poxvirus, and adenovirus.
Baculoviruses encode proteins that are termed inhibitors of apoptosis (IAPs) because they inhibit the apoptosis that would otherwise occur when insect cells are infected by the virus. These proteins are thought to work in a manner that is independent of other viral proteins. The baculovirus IAP genes include sequences encoding a ring zinc finger-like motif (RZF), which is presumed to be directly involved in DNA binding, and two N-terminal domains that consist of a 70 amino acid repeat motif termed a BIR domain (Baculovirus IAP Repeat).
The role of apoptosis in cancer has only recently been appreciated. The identification of growth promoting xe2x80x9concogenesxe2x80x9d in the late 1970""s gave rise to an almost universal focus on cellular proliferation that dominated research in cancer biology for many years. Long-standing dogma held that anti-cancer therapies preferentially targeted rapidly dividing cancer cells relative to xe2x80x9cnormalxe2x80x9d cells. This explanation was not entirely satisfactory, since some slow growing tumors are easily treated, while many rapidly dividing tumor types are extremely resistant to anti-cancer therapies. Progress in the cancer field has now led to a new paradigm in cancer biology wherein neoplasia is viewed as a failure to execute normal pathways of programmed cell death. Normal cells receive continuous feedback from their neighbors through various growth factors, and commit xe2x80x9csuicidexe2x80x9d if removed from this context. Cancer cells somehow ignore these commands and continue inappropriate proliferation. Cancer therapies, including radiation and many chemotherapies, have traditionally been viewed as causing overwhelming cellular injury. New evidence suggests that cancer therapies actually work by triggering apoptosis.
Both normal cell types and cancer cell types display a wide range of susceptibility to apoptotic triggers, although the determinants of this resistance are only now under investigation. Many normal cell types undergo temporary growth arrest in response to a sub-lethal dose of radiation or cytotoxic chemical, while cancer cells in the vicinity undergo apoptosis. This provides the crucial treatment xe2x80x9cwindowxe2x80x9d of appropriate toxicity that allows successful anti-cancer therapy. It is therefore not surprising that resistance of tumor cells to apoptosis is emerging as a major category of cancer treatment failure.
Compared to the numerous growth-promoting oncogenes identified to date ( greater than 100), relatively few genes have been isolated that regulate apoptosis. The Bcl-2 gene was first identified as an oncogene associated with the development of follicular lymphomas. In contrast to all other oncogenes identified to date, Bcl-2 displays no ability to promote cell proliferation, and instead has been demonstrated to suppress apoptosis by a variety of triggers. Elevated Bcl-2 expression is associated with a poor prognosis in neuroblastoma, prostate and colon cancer, and can result in a multidrug resistant phenotype in vitro. Although the study of Bcl-2 has helped revolutionize cancer paradigms, the vast majority of human malignancies do not demonstrate aberrant Bcl-2 expression.
In contrast to the findings with Bcl-2, mutation of the p53 tumor suppresser gene has been estimated to occur in up to 50% of human cancers and is the most frequent genetic change associated with cancer to date. The p53 protein plays a crucial role in surveying the genome for DNA damage. The cell type and degree of damage determines whether the cell will undergo growth arrest and repair, or initiate apoptosis. Mutations in p53 interfere with this activity, rendering the cell resistant to apoptosis by a wide range of cellular insults. Some progress has been made in understanding the molecular biology of p53, but many questions remain. p53 is known to function as a transcription factor, with the ability to positively or negatively regulate the expression of a variety of genes involved in cell cycle control, DNA repair, and apoptosis (including the anti-apoptotic Bcl-2 gene described above and the related pro-apoptotic gene Bax). The drug resistant phenotype conferred by p53 alterations has been linked to Bcl-2/Bax regulation, but this correlation does not hold for most cancer types, leaving open the possibility that other critical genes regulated by p53 remain to be identified.
We have discovered that inhibitor of apoptosis (IAP) protein overexpression is associated with a wide range of cancer types including ovarian cancer, adenocarcinoma, lymphoma, and pancreatic cancer. In addition, we have found that nuclear localization, fragmentation of the IAPs, and overexpression of the IAPs in the presence of p53 mutations correlate with a cancer diagnosis, a poor prognosis, and resistance to numerous chemotherapeutic cancer drugs. These discoveries provide diagnostic, prognostic, and therapeutic compounds and methods for the detection and treatment of proliferative diseases. One way in which the expression of an IAP in a cell can be decreased is by administering to the cell a negative regulator of the IAP apoptotic pathway, for example, an antisense nucleic acid.
In general, the invention features methods and reagents useful for inducing apoptosis in a cell. The methods and reagents of the invention are useful in treating cancers, and other proliferative diseases.
In a first aspect, the invention features an inhibitor of apoptosis (IAP) antisense nucleic acid that inhibits IAP biological activity, regardless of the length of the antisense nucleic acid. In preferred embodiments, the IAP is XIAP, HIAP1, or HIAP2. In other preferred embodiments, the antisense nucleic acid is mammalian, for example, mouse or human. In yet another embodiment, the antisense nucleic acid is between 8 and 30 nucleotides in length.
In still other further preferred embodiments, the XIAP antisense is chosen from any one of SEQ ID NOS: 1 through 96, and the HIAP1 antisense is chosen from any one of SEQ ID NOS: 97 through 194. Preferably the IAP biological activity is inhibition of apoptosis or inhibition of IAP RNA or polypeptide expression. The antisense nucleic acid may comprise at least one modified internucleoside linkage. Preferably the modified internucleoside linkage is a phosphorothioate, a methylphosphonate, a phosphotriester, a phosphorodithioate, or a phosphoselenate linkage. In addition, the antisense nucleic acid may comprise at least one modified sugar moiety. Preferably this modified sugar moiety is a 2xe2x80x2-O methoxyethyl group or a 2xe2x80x2-O methyl group. In still another preferred embodiment, the antisense nucleic acid is a chimeric nucleic acid. Preferably the chimeric nucleic acid comprises DNA residues linked together by phosphorothioate linkages, and the DNA residues are flanked on each side by at least one 2xe2x80x2-O methyl RNA residue linked together by a phosphorothioate linkage. More preferably the DNA residues are flanked on each side by at least three 2xe2x80x2-O methyl RNA residues. In yet another embodiment, the antisense nucleic acid is a ribozyme.
In a second aspect, the invention features a method of enhancing apoptosis in a cell, involving administering to the cell a negative regulator of the IAP-dependent antiapoptotic pathway. In preferred embodiments the negative regulator is an antisense IAP nucleic acid, an antibody that specifically binds an IAP polypeptide, an IAP polypeptide comprising a ring zinc finger, said polypeptide having no more than two BIR domains, a nucleic acid encoding the ring zinc finger domain of an IAP polypeptide, or a compound that prevents cleavage of the IAP polypeptide.
In preferred embodiments of the second aspect of the invention, the cell is in a mammal diagnosed with a proliferative disease, for example, cancer, The cell may comprises a mucosa-associated lymphoid tissue (MALT), a tissue in which the IAP gene HIAP1 is frequently involved in a translocation, resulting in marginal zone cell lymphomas. The cell may also be a breast cancer cell, where increased HIAP1 expression is known to correlate with tumor progression. The cell may also be a cell in which NFkB expression or activity is increased, for example, cell of head and neck carcinomas, adult T-cell lymphomas, nasopharyngeal carcinomas, and Hodgkin""s disease. The cell may also be an acute myelogenous leukemia cell, where increased XIAP levels correlate with poor patient prognosis. In addition, the cell may be a small cell lung carcinoma cell, where increased levels of XIAP correlates with increased resistance to radiation treatment.
In preferred embodiments of the second aspect of the invention, the IAP is XIAP, HIAP1, or HIAP2. Preferably the antisense nucleic acid is mammalian, for example, mouse or human. In still other preferred embodiments, the XIAP antisense is chosen from any one of SEQ ID NOS: 1 through 96, and the HIAP1 antisense is chosen from any one of SEQ ID NOS: 97 through 194.
In still other embodiments of the second aspect of the invention, the antisense nucleic acid comprises at least one modified internucleoside linkage. Preferably the modified internucleoside linkage is a phosphorothioate, a methylphosphonate, a phosphotriester, a phosphorodithioate, or a phosphoselenate linkage. In addition, the antisense nucleic acid may comprise at least one modified sugar moiety. Preferably this modified sugar moiety is a 2xe2x80x2-O methoxyethyl group or a 2xe2x80x2-O methyl group. In still another preferred embodiment, the antisense nucleic acid is a chimeric nucleic acid. Preferably the chimeric nucleic acid comprises DNA residues linked together by phosphorothioate linkages, and the DNA residues are flanked on each side by at least one 2xe2x80x2-O methyl RNA residue linked together by a phosphorothioate linkage. More preferably the DNA residues are flanked on each side by at least three 2xe2x80x2-O methyl RNA residues. In still further embodiments, administration of the antisense nucleic acid sensitizes the cell to chemotherapy or radiotherapy. In addition, the cell may be in vitro or in vivo.
In a third aspect, the invention features a pharmaceutical composition comprising a mammalian IAP antisense nucleic acid. In one preferred embodiment, the mammalian antisense IAP nucleic acid is a human antisense nucleic acid. Preferably the antisense nucleic acid binds a target sequence of the human XIAP gene or mRNA, the human HIAP1 gene or mRNA, the human HIAP2 gene or mRNA, the murine XIAP gene or mRNA, the murine HIAP1 gene or mRNA, or the murine HIAP2 gene or mRNA. More preferably the composition comprises an antisense nucleic acid chosen from any one of SEQ ID NOS: 1 through 96 (XIAP) or SEQ ID NOS: 97 through 194 (HIAP1).
In another aspect, the invention features an IAP gene nucleic acid fragment or antisense RNA sequence for use in suppressing cell proliferation. Such nucleic acids of the invention and methods for using them may be identified according to a method involving: (a) providing a cell sample; (b) introducing by transformation into the cell sample a candidate IAP nucleic acid; (c) expressing the candidate IAP nucleic acid within the cell sample; and (d) determining whether the cell sample exhibits an altered apoptotic response, whereby decreased apoptosis identifies an anti-proliferative compound. Preferably the cell is a cancer cell.
In another aspect, the invention features a method of treating a patient diagnosed with a proliferative disease. In the method, apoptosis may be induced in a cell to control a proliferative disease either alone or in combination with other therapies by administering to the cell a negative regulator of the IAP-dependent or anti-apoptotic pathway. The negative regulator may be, but is not limited to, an IAP ring zinc finger, and an IAP polypeptide that includes a ring zinc finger and lacks at least one BIR domain. Alternatively, apoptosis may be induced in the cell by administering a nucleic acid encoding an IAP antisense RNA molecule administered directly or via gene therapy (see U.S. Pat. No. 5,576,208 for general parameters that may be applicable in the selection of IAP antisense RNAs). In yet another method, the negative regulator may be a purified antibody, or a fragment thereof, that binds specifically to an IAP polypeptide. For example, in one preferred embodiment, the antibody may bind to an approximately 26 kDa cleavage product of an IAP polypeptide that includes at least one BIR domain but lacks a ring zinc finger domain.
In two additional aspects, the invention features a transgenic animal and methods of using the mammal for detection of anti-cancer therapeutics. Preferably the mammal overexpresses an IAP polypeptide and/or expresses an IAP antisense RNA or IAP fragment. In one embodiment, the animal also has a genetic predisposition to cancer or has cancer cells under conditions that provide for proliferation absent the transgenic construct encoding either the antisense RNA or fragment.
xe2x80x9cProteinxe2x80x9d or xe2x80x9cpolypeptidexe2x80x9d or xe2x80x9cpolypeptide fragmentxe2x80x9d means any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally-occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
xe2x80x9cApoptosisxe2x80x9d means the process of cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cell membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering. Cells that die by apoptosis include neurons (e.g., during the course of neurodegenerative diseases such as stroke, Parkinson""s disease, and Alzheimer""s disease), cardiomyocytes (e.g., after myocardial infarction or over the course of congestive heart failure), and cancer cells (e.g., after exposure to radiation or chemotherapeutic agents). Environmental stress (e.g., hypoxic stress) that is not alleviated may cause a cell to enter the early phase of the apoptotic pathway, which is reversible (i.e., cells at the early stage of the apoptotic pathway can be rescued). At a later phase of apoptosis (the commitment phase), cells cannot be rescued, and, as a result, are committed to die.
Proteins and compounds known to stimulate and inhibit apoptosis in a diverse variety of cells are well known in the art. For example, intracellular expression and activation of the caspase (ICE) family induces or stimulates apoptotic cell death, whereas expression of the IAPs or some members of the Bcl-2 family inhibits apoptotic cell death. In addition, there are survival factors that inhibit cell death in specific cell types. For example, neurotrophic factors, such as NGF inhibit neuronal apoptosis.
In some situations it may be desirable to artificially stimulate or inhibit apoptotic cell death by gene therapy or by a compound that mimics a gene therapeutic effect. For example, a cell that is susceptible to apoptosis induced by disease or environmental stress may be made more resistant to apoptosis by introducing an expression vector encoding an anti-apoptotic protein (such as an IAP, a Bcl-2 family member, or a neurotrophin) into the cell. Conversely, a cancer cell may be made less resistant to apoptosis by introducing into it an expression vector encoding a pro-apoptotic protein (such as a caspase) or by introducing into it an antisense nucleic acid, for example, an IAP antisense nucleic acid, regardless of its length. In addition, placement of the encoded protein of interest under the translational regulation of a XIAP IRES ensures that copious quantities of the protein are produced, especially under cellular conditions during which most protein translation (i.e., cap-dependent protein translation) is down-regulated, e.g., when a cell is under environmental stress, and when a cell is at a threshold for entering the apoptotic pathway.
By xe2x80x9cIAP genexe2x80x9d is meant a gene encoding a polypeptide having at least one BIR domain and a ring zinc finger domain that is capable of modulating (inhibiting or enhancing) apoptosis in a cell or tissue when provided by other intracellular or extracellular delivery methods (see, e.g., U.S. Pat. No. 5,919,912, U.S. Ser. No. 08/576,965, and PCT/IB96/01022). In preferred embodiments, the IAP gene is a gene having about 50% or greater nucleotide sequence identity to at least one of the IAP amino acid encoding sequences of FIGS. 1 through 6, or portions thereof. Preferably the region of sequence over which identity is measured is a region encoding at least one BIR domain and a ring zinc finger domain. Mammalian IAP genes include nucleotide sequences isolated from any mammalian source. Preferably the mammal is a human.
The term xe2x80x9cIAP genexe2x80x9d is meant to encompass any member of the family of genes that encode inhibitors of apoptosis. An IAP gene may encode a polypeptide that has at least 20%, preferably at least 30%, and most preferably at least 50% amino acid sequence identity with at least one of the conserved regions of one of the IAP members described herein (i.e., either the BIR or ring zinc finger domains from human or murine XIAP, HIAP1, and HIAP2). Representative members of the IAP gene family include, without limitation, the human and murine XIAP, HIAP1, and HIAP2 genes.
By xe2x80x9cIAP proteinxe2x80x9d or xe2x80x9cIAP polypeptidexe2x80x9d is meant a polypeptide, or fragment thereof, encoded by an IAP gene.
By xe2x80x9cBIR domainxe2x80x9d is meant a domain having the amino acid sequence of the consensus sequence: Xaa1-Xaa1-Xaa1-Arg-Leu-Xaa1-Thr-Phe-Xaa1-Xaa1-Trp-Pro-Xaa2-Xaa1-Xaa1-Xaa2-Xaa2-Xaa1-Xaa1-Xaa1-Xaa1-Leu-Ala-Xaa1-Ala-Gly-Phe-Tyr-Tyr-Xaa1-Gly-Xaa1-Xaa1-Asp-Xaa1-Val-Xaa1-Cys-Phe-Xaa1-Cys-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Trp-Xaa1-Xaa1-Xaa1-Asp-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-His-Xaa1-Xaa1-Xaa1-Xaa1-Pro-Xaa1-Cys-Xaa1-Phe-Val, wherein Xaal is any amino acid and Xaa2 is any amino acid or is absent (SEQ ID NO: 216). Preferably the sequence is substantially identical to one of the BIR domain sequences provided for XIAP, HIAP 1, or HIAP2 herein.
By xe2x80x9cring zinc fingerxe2x80x9d or xe2x80x9cRZFxe2x80x9d is meant a domain having the amino acid sequence of the consensus sequence: Glu-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa2-Xaa1-Xaa1-Xaa1-Cys-Lys-Xaa3-Cys-Met-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Xaa3-Xaa1-Phe-Xaa1-Pro-Cys-Gly-His-Xaa1-Xaa1-Xaa1-Cys-Xaa1-Xaa1-Cys-Ala-Xaa1-Xaa1-Xaa1-Xaa1-Xaa1-Cys-Pro-Xaa1-Cys, wherein Xaa1 is any amino acid, Xaa2 is Glu or Asp, and Xaa3 is Val or lie (SEQ ID NO: 217).
Preferably the sequence is substantially identical to the RZF domains provided in U.S. Ser. No. 08/800,929, incorporated herein by reference, for the human or murine XIAP, HIAP1, or HIAP2.
By xe2x80x9cenhancing apoptosisxe2x80x9d is meant increasing the number of cells that apoptose in a given cell population. Preferably the cell population is selected from a group including ovarian cancer cells, breast cancer cells, pancreatic cancer cells, T cells, neuronal cells, fibroblasts, or any other cell line known to proliferate in a laboratory setting. It will be appreciated that the degree of apoptosis enhancement provided by an apoptosis-enhancing compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis that identifies a compound that enhances apoptosis otherwise limited by an IAP. Preferably xe2x80x9cenhancing apoptosisxe2x80x9d means that the increase in the number of cells undergoing apoptosis is at least 25%, more preferably the increase is 50%, and most preferably the increase is at least one-fold. Preferably the sample monitored is a sample of cells that normally undergo insufficient apoptosis (i.e., cancer cells). Methods for detecting a changes in the level of apoptosis (i.e., enhancement or reduction) are described herein.
By xe2x80x9cproliferative diseasexe2x80x9d is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancers such as lymphoma, leukemia, melanoma, ovarian cancer, breast cancer, pancreatic cancer, and lung cancer are all examples of proliferative disease.
By xe2x80x9cIAP biological activityxe2x80x9d is meant any activity known to be caused in vivo or in vitro by an IAP polypeptide.
By xe2x80x9ctransformed cellxe2x80x9d is meant a cell into which (or into an ancestor of which) has been introduced, by means of recombinant DNA techniques, a DNA molecule encoding (as used herein) an IAP polypeptide.
By xe2x80x9ctransgenexe2x80x9d is meant any piece of DNA that is inserted by artifice into a cell, and becomes part of the genome of the organism that develops from that cell. Such a transgene may include a gene that is partly or entirely heterologous (i.e., foreign) to the transgenic organism, or may represent a gene homologous to an endogenous gene of the organism.
By xe2x80x9ctransgenicxe2x80x9d is meant any cell that includes a DNA sequence that is inserted by artifice into a cell and becomes part of the genome of the organism that develops from that cell. As used herein, the transgenic organisms are generally transgenic mammals (e.g., rodents, such as rats or mice) and the DNA (transgene) is inserted by artifice into the nuclear genome.
By xe2x80x9ctransformationxe2x80x9d is meant any method for introducing foreign molecules, for example, an antisense nucleic acid, into a cell. Lipofection, calcium phosphate precipitation, retroviral delivery, electroporation, biolistic transformation, and penetratin are just a few of the teachings that may be used. For example, biolistic transformation is a method for introducing foreign molecules into a cell using velocity driven microprojectiles such as tungsten or gold particles. Such velocity-driven methods originate from pressure bursts that include, but are not limited to, helium-driven, air-driven, and gunpowder-driven techniques. Biolistic transformation may be applied to the transformation or transfection of a wide variety of cell types and intact tissues including, without limitation, intracellular organelles (e.g., and mitochondria and chloroplasts), bacteria, yeast, fungi, algae, animal tissue, and cultured cells. In another example, a foreign molecule (e.g., an antisense nucleic acid) can be translocated into a cell using the penetratin system as described, for example, by Prochiantz (Nature Biotechnology 16:819-820, 1998; and Derossi et al. (Trends Cell Biol. 8: 84-87, 1998). In this system a penetratin peptide contains a transduction sequence that carries the peptide and a conjugated partner, for example, a phosphorothioate antisense nucleic acid (that is cross-linked through a disulfide bridge to the peptide) across the plasma membrane into the cell. The disulfide band is reduced inside the cell, releasing the partner.
By xe2x80x9cantisense,xe2x80x9d as used herein in reference to nucleic acids, is meant a nucleic acid sequence, regardless of length, that is complementary to the coding strand or mRNA of an IAP gene. Preferably the antisense nucleic acid is capable of enhancing apoptosis when present in a cell that normally does not undergo sufficient apoptosis. Preferably the increase is at least 10%, relative to a control, more preferably 25%, and most preferably 1-fold or more. Preferably an IAP antisense nucleic acid comprises from about 8 to 30 nucleotides. An IAP antisense nucleic acid may also contain at least 40, 60, 85, 120, or more consecutive nucleotides that are complementary to a IAP mRNA or DNA, and may be as long as a full-length IAP gene or mRNA. The antisense nucleic acid may contain a modified backbone, for example, phosphorothioate, phosphorodithioate, or other modified backbones known in the art, or may contain non-natural internucleoside linkages.
By xe2x80x9cribozymexe2x80x9d is meant an RNA that has enzymatic activity, possessing site specificity and cleavage capability for a target RNA molecule. Ribozymes can be used to decrease expression of a polypeptide. Methods for using ribozymes to decrease polypeptide expression are described, for example, by Turner et al., (Adv. Exp. Med. Biol. 465:303-318, 2000) and Norris et al., (Adv. Exp. Med. Biol. 465:293-301, 2000).
By xe2x80x9csubstantially identicalxe2x80x9d is meant a polypeptide or nucleic acid exhibiting at least 50%, preferably 85%, more preferably 90%, and most preferably 95% homology to a reference amino acid or nucleic acid sequence. For polypeptides, the length of comparison sequences will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids. For nucleic acids, the length of comparison sequences will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 110 nucleotides.
Sequence identity is typically measured using sequence analysis software with the default parameters specified therein (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). This software program matches similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine, valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
By xe2x80x9csubstantially pure polypeptidexe2x80x9d is meant a polypeptide that has been separated from the components that naturally accompany it. Typically, the polypeptide is substantially pure when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably the polypeptide is an IAP polypeptide that is at least 75%), more preferably at least 90%, and most preferably at least 99%, by weight, pure. A substantially pure IAP polypeptide may be obtained, for example, by extraction from a natural source (e.g., a fibroblast, neuronal cell, or lymphocyte) by expression of a recombinant nucleic acid encoding an IAP polypeptide, or by chemically synthesizing the protein. Purity can be measured by any appropriate method, e.g., by column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
A protein is substantially free of naturally associated components when it is separated from those contaminants that accompany it in its natural state. Thus, a protein that is chemically synthesized or produced in a cellular system different from the cell from which it naturally originates will be substantially free from its naturally associated components. Accordingly, substantially pure polypeptides include those derived from eukaryotic organisms but synthesized in E. coli or other prokaryotes.
By xe2x80x9csubstantially pure DNAxe2x80x9d is meant DNA that is free of the genes that, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cpositioned for expressionxe2x80x9d is meant that the DNA molecule is positioned adjacent to a DNA sequence, that directs transcription and translation of the sequence (i.e., facilitates the production of, e.g., an IAP polypeptide, a recombinant protein or an RNA molecule).
By xe2x80x9creporter genexe2x80x9d is meant a gene whose expression may be assayed; such genes include, without limitation, glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and Beta-galactosidase.
By xe2x80x9cpromoterxe2x80x9d is meant a minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements that are sufficient to render promoter-dependent gene expression controllable for cell type-specific, tissue-specific or that are inducible by external signals or agents; such elements may be located in the 5xe2x80x2 or 3xe2x80x2 regions of the native gene.
By xe2x80x9coperably linkedxe2x80x9d is meant that a gene and one or more regulatory sequences are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequences.
By xe2x80x9cconserved regionxe2x80x9d is meant any stretch of six or more contiguous amino acids exhibiting at least 30%, preferably 50%, and most preferably 70% amino acid sequence identity between two or more of the IAP family members, (e.g., between human HIAP1, HIAP2, and XIAP). Examples of preferred conserved regions include, without limitation, BIR domains and ring zinc finger domains.
By xe2x80x9cdetectably-labelledxe2x80x9d is meant any means for marking and identifying the presence of a molecule, e.g., an oligonucleotide probe or primer, a gene or fragment thereof, or a cDNA molecule. Methods for detectably-labelling a molecule are well known in the art and include, without limitation, radioactive labelling (e.g., with an isotope such as 32P or 35S) and nonradioactive labelling (e.g., chemiluminescent labeling or fluorescein labelling).
By xe2x80x9cpurified antibodyxe2x80x9d is meant an antibody that is at least 60%, by weight, free from proteins and naturally occurring organic molecules with which it is naturally associated. Preferably the preparation is at least 75, more preferably 90%, and most preferably at least 99%, by weight, antibody, e.g., an IAP-specific antibody. A purified antibody may be obtained, for example, by affinity chromatography using recombinantly-produced protein or conserved motif peptides and standard techniques.
By xe2x80x9cspecifically bindsxe2x80x9d is meant an antibody that recognizes and binds a protein but that does not substantially recognize and bind other molecules in a sample, e.g., a biological sample, that naturally includes protein.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.